Bottom Line:
This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

ABSTRACTCysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

Figure 3: zVAD-CH2F inhibits the processing of CPP32 and the cleavage of PARP. CPP32 activation and cleavage of PARP in thymocytes cultured with dexamethasone for 6 h in the presence or absence of 100 μM zVAD-CH2F. Cell lysates were electrophoresed, and substrate cleavage was visualized by immunoblotting as described in Materials and Methods.

Mentions:
The tripeptide zVAD-CH2F is a well known inhibitor of cysteine proteases of ICE/CED-3 family, including CPP32 (reference 23 and Fig. 3). To confirm the involvement of CPP32-like activity in spontaneous apoptosis, the inhibition profile of the tripeptide on thymocyte cultures was determined. The results presented in Fig. 4 are representative of two independent experiments. Interestingly, zVAD-CH2F was a potent inhibitor of spontaneous apoptosis of thymocytes as well as apoptosis mediated either by anti-CD3 mAbs or dexamethasone. Altogether, these results confirm the direct involvement of CPP32 and CPP32-like proteases in thymocyte apoptosis.

Figure 3: zVAD-CH2F inhibits the processing of CPP32 and the cleavage of PARP. CPP32 activation and cleavage of PARP in thymocytes cultured with dexamethasone for 6 h in the presence or absence of 100 μM zVAD-CH2F. Cell lysates were electrophoresed, and substrate cleavage was visualized by immunoblotting as described in Materials and Methods.

Mentions:
The tripeptide zVAD-CH2F is a well known inhibitor of cysteine proteases of ICE/CED-3 family, including CPP32 (reference 23 and Fig. 3). To confirm the involvement of CPP32-like activity in spontaneous apoptosis, the inhibition profile of the tripeptide on thymocyte cultures was determined. The results presented in Fig. 4 are representative of two independent experiments. Interestingly, zVAD-CH2F was a potent inhibitor of spontaneous apoptosis of thymocytes as well as apoptosis mediated either by anti-CD3 mAbs or dexamethasone. Altogether, these results confirm the direct involvement of CPP32 and CPP32-like proteases in thymocyte apoptosis.

Bottom Line:
This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

ABSTRACTCysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.